PROJECT SUMMARY Approximately 29,000 men in the United States were estimated to die from prostate cancer in 2018. As our current therapies are not working for a large number of men with metastatic castration-resistant prostate cancer, we need innovative strategies to identify new therapeutic approaches. Advances in dissecting mechanisms of metabolic reprogramming in several tumor types has led to widespread interest in drugging metabolic vulnerabilities as a therapeutic strategy for cancer. Defining the factors that regulate metabolic reprogramming in prostate cancer is fundamental to understanding prostate cancer pathogenesis and likely to yield therapeutic targets for this lethal disease. Two key factors that regulate metabolic reprogramming in cancer and inform metabolic vulnerabilities that can be targeted for therapy are the disease-initiating oncogenes and the tissue or cells-of-origin. However, it is not well understood how the cells-of-origin for prostate cancer cooperate with oncogenes to reprogram metabolism. Using genetically engineered mouse models and human transformation assays, we and others have demonstrated that both prostate basal and luminal cells can serve as target cells for prostate transformation. Genetically altered basal cells must differentiate into luminal cells in order to initiate cancer, establishing a key role for differentiation in basal cell-initiated tumorigenesis. In response to hormonal therapy, luminal-like prostate cancer cells undergo lineage plasticity and exhibit basal cell features, demonstrating a role for differentiation in disease progression and therapy-resistance. Surprisingly, little is known about (1) metabolic activity in distinct epithelial cells of the prostate, (2) how changes in metabolism regulate epithelial differentiation, and (3) how oncogenic transformation reprograms prostate epithelial metabolism. Understanding prostate cancer metabolism requires first understanding prostate epithelial metabolism. However, such data on the metabolism of primary cell-types does not yet exist. In this proposal, mouse and human tissue will be studied in parallel to determine the extent to which the cells-of-origin and the disease-initiating oncogenes regulate metabolic reprogramming in prostate cancer. In Aim 1, we will establish metabolic signatures of distinct cells-of-origin for prostate cancer (basal and luminal). In Aim 2, we will evaluate metabolic regulation of luminal differentiation, a requirement for prostate tumorigenesis. In Aim 3, we will determine how epithelial metabolism is reprogrammed in response to genetic alterations. This novel approach will enhance our fundamental understanding of prostate tumorigenesis and may lead to new therapeutic strategies to combat lethal prostate cancer.